In Acanthamoeba castellanii lysosomal enzymes are secreted and accumulate in the growth medium. We have previously shown that the secretion rates of eight enzymes fall into two groups: one about 17% of total cell activity per hour and another at about 3%. The lysosomal enzymes appear to be secreted coincident with membrane recycling from the digestive vacuoles. In order to obtain markers to trace the secretion route, we have isolated and partially purified one enzyme from each group, beta-D-hexosaminidase from the high secretory rate group, and beta-D-glucosidase from the low secretory rate group. Hexosaminidase, purified to near homogeneity by a combination of ion exchange and affinity chromatography, has a isoelectric point of 5.82 and an apparent subunit molecular weight of 52 kDa. Beta- glucosidase has been partially purified, has an isoelectric point of 5.2 and an apparent molecular weight of 105 kDa. The deglycosylated, purified enzymes will be used to prepare antibodies for immunocytochemical studies of the membrane recyling and enzyme secretion route.